The present invention relates generally to the quantitation of human and murine laminin mRNA, as well as human and murine .beta.-actin mRNA.
The polymerase chain reaction (PCR) makes possible the amplification of specific segments of nucleic acids via in vitro enzymatic synthesis and is thus a powerful tool for the detection of particular gene transcripts where standard methods may be limited by the small amount of starting tissue available or by the low abundance of transcripts. In the PCR technique, short oligonucleotide primers are synthesized to match opposite ends of the nucleic acid sequence to be amplified. The target sequence flanked by the primers need not be known. In the first PCR cycle, a sample of DNA or RNA (converted to cDNA prior to use in the PCR technique) is extracted from a specimen, denatured, and hybridized with the primers, which are present in molar excess. A polymerase catalyzes the duplication of the target sequence by the addition of deoxynucleotide triphosphates (dNTPs) to the primers at their 3' ends. The dNTPs attached to the primers complement the nucleic acid sequence of the target and thus duplicate the sequence of one strand of the original DNA.
In the second step of the cycle, the replicated DNA is denatured, hybridized with the oligonucleotide primers, and returned to polymerization conditions to again duplicate the target sequence. This sequence is then repeated, and the repetitive cycle of primer annealing, primer extension, and denaturation of the template/PCR product results in rapid, exponential, and voluminous amplification of specific segments of DNA or RNA. Thus, the PCR process is useful for detection of small amounts of nucleic acid or for acquiring sufficient quantities of DNA or RNA for research. See U.S. Pat. Nos. 4,683,195 and 4,800,159, the respective contents of which are incorporated herein by reference.
Compared with other detection methods, such as northern blot analysis, PCR provides a more sensitive and accurate method for detecting the presence of relatively small quantities of DNA or RNA. PCR has also been used to quantify small quantities of mRNA. Thus, Wang et al., Proc. Nat'l Acad. Sci. (U.S.A.) 86: 9717-21 (1989), disclose using PCR and a DNA control template to quantify lymphokine mRNA from samples as small as 1 ng. Laminin is an extracellular matrix glycoprotein consisting of three polypeptide chains, A, B1 and B2, each coded for by a separate gene. In contrast, .beta.-actin is an intracellular protein and consists of only one polypeptide chain. Also, the .beta.-actin gene is regulated differently than are the laminin genes.
The expression of laminin is of extreme interest due to the role of laminin in diverse biological activities such as the induction of cellular adhesion, differentiation, migration, and growth of many cell types. For example, laminin is involved in the development of normal kidney and its disregulation contributes to glomerulosclerosis in renal disease.
Lack of sufficient biopsy material containing laminin and .beta.-actin genes combined with low transcription level of these genes have hindered the study of the expression of these genes. As noted above, PCR provides a potential avenue for detecting and quantifying the expression of genes at the transcriptional level. Using PCR to monitor expression of the laminin and .beta.-actin genes, however, poses a difficult task at best. For example, the researcher must use the appropriate primers and reaction parameters because it is known that certain reaction parameters that function efficiently for amplification with one set of primers may not do so for another set. Moreover, primers may cross hybridize to non-target sequences. In addition, quantitating the amount of amplification which occurs during one complete PCR reaction is difficult due to different efficiencies in annealing rates of different primers and non-linear amplification of target sequences and control sequences.